Regulation of expression of the DNA repair gene-O⁶-methylguanine-DNA methyltransferase via protein kinase C-mediated signaling

O⁶-akylguanine is the major mutagenic and cytotoxic DNA lesion induced by alkylating agents, including 2-chloroethyl-N-nitrosaurea-based antitumor drugs. This lesion is repaired by O⁶-methylguanine-DNA methyltransferase (MGMT), the expression of which is highly variable in both normal tissues and in tumor cells. The promoter of the human MGMT gene was found to contain two putative activator protein (AP)-1 sites. Here, we show that the level of MGMT mRNA in HeLa S3 cells was increased 3-5-fold by phorbol-12-myristate-13- acetate (TPA) and 1,2-diacyl-sn-glycerol (DAG), which are activators of protein kinase C(PKC), as well as by okaidic acid, an inhibitor of protein phosphatases. The PKC inhibitor 1-(5-isoquinoline sulfonyl)-2- methylpiperazine-HCl eliminated MGMT activation by TPA and DAG but not by OA. Prior down-regulation of PKC abolished subsequent effects of TPA or DAG. The results indicate AP-1 to be involved in regulation of MGMT expression. This hypothesis was supported by showing AP-1 binding to two target sequences of the MGMT promoter and transactivation of the MGMT promoter upon cotransfection with c-fos and c-jun in F9 cells. That TPA-mediated induction of MGMT caused increased cellular resistance to 2-chloroethyl-N-nitrosaurea suggest a therapeutic significance for PKC-mediated MGMT modulation.